The effect of bacterial peptidoglycans on the immune response of hamsters to influenza virus vaccines

The immune response of hamsters to influenza virus vaccine incorporating peptidoglycans derived from Streptomyces griseus was investigated. The results showed that although inoculation of a subunit influenza virus vaccine together with a peptidoglycan elicited a markedly increased serum HI antibody response compared to that induced by the subunit vaccine alone, pre-treatment of animals with peptidoglycans resulted in a suppression of the antibody response to the vaccine haemagglutinin antigen. The immunosuppressive effect was dose-related, and could be shown by adoptive transfer experiments to be transferred by spleen cell preparations from peptidoglycan-treated hamsters. The reasons for these findings, and their implication for the use of peptidoglycans as 'carriers' in influenza virus subunit vaccines, are discussed.     

The immune response of hamsters to purified haemagglutinins and whole influenza virus vaccines following live influenza virus infection

The ability of several, live type A influenza viruses to enhance the serum haemagglutination-inhibiting (HI) antibody response of hamsters to subsequent immunization with inactivated, heterotypic influenza virus vaccines was examined. Live influenza viruses were found to vary in their priming ability for a given vaccine, and a given virus was not able to prime for all inactivated vaccines to an equal extent. Common determinants in the haemagglutinin antigens of the priming virus and the vaccine virus were suggested as responsible for the enhancement of the antibody response to some of the vaccines, but for other pairs of viruses the haemagglutinin antigens were distinct. Thus, enhancement in these instances cannot be due to cross-reacting haemagglutinins. Pre-infection of hamsters by several influenza type A viruses was employed in an attempt to enhance the serum HI antibody response to purified, haemagglutinin antigens prepared from A/PR/8/34 and the MRC-2 recombinant strain of A/England/42/72 viruses. Although prior infection enhanced the antibody response to whole virus, this was not demonstrable for the purified haemagglutinin components of the virus. The possible reasons for this are discussed.     

Immunity to influenza virus infection induced by heterologous,inactivated vaccines

The ability of inactivated influenza A vaccines to induce serum HI antibody and immunity to challenge infection was studied in hamsters and in volunteers. Groups of hamsters were immunized with 200 IU of influenza virus A/Scotland/74, A/Port Chalmers/73, A/England/72, or A/Hong Kong/68. The serum HI antibody response of animals to, and immunity to challenge infection was directly related to the known relationship between the vaccine and test viruses. Thus, hamsters given A/Hong Kong/68 or A/England/72 vaccine produced serum HI antibody and immunity to A/Hong Kong virus infection, and animals given A/Scotland/74, A/Port Chalmers/73, and A/England/72 produced antibody and immunity to A/Scotland infection.In a volunteer study, groups of students were immunized with 400 IU of the same vaccines as used above. The ability to infect these volunteers with WRL 105 virus given 4 weeks later was directly related to the vaccine-induced serum HI antibody to the challenge virus. The highest titers of serum HI antibody to A/Scotland virus were found in volunteers inoculated with homologous vaccine, lower titers were found in volunteers given A/Port Chalmers or A/England/ 72 vaccine and the lowest levels were seen in volunteers given A/Hong Kong/68 vaccine: the largest number of infections by the challenge virus was seen in volunteers given A/Hong Kong/68 vaccine, less were observed in volunteers given A/England/72 vaccine, and least were found in groups given A/Port Chalmers or A/Scotland/74 vaccine. Compared with the incidence of infection in volunteers given B/Hong Kong/73 vaccine, all groups given heterologous influenza A vaccines showed some immunity to challenge infection.     

Immunity to Influenza in Ferrets: II. Influence of Adjuvants on Immunization

Immunization of ferrets with a single intramuscular inoculation of killed A2/Hong Kong virus did not induce serum or nasal antibody, and these animals were found to be completely susceptible to subsequent infection with virulent influenza virus A2/Hong Kong/3/68. A similar result was found for ferrets immunized with 2 inoculations of killed virus vaccine given 2 weeks apart. Ferrets immunized with killed A2/Hong Kong virus in conjunction with Bordetella pertussis produced relatively low levels of serum HI antibody to A2/Hong Kong virus; when infected with virulent influenza virus, these ferrets showed a modified reaction, with a less marked febrile reaction than was observed for non-immunized animals.Immunization of ferrets with killed A2/Hong Kong virus in Freund''s complete adjuvant resulted in the production of relatively high levels of serum HI antibody, but no detectable nasal antibody. These animals were shown to be partially immune to subsequent infection with virulent influenza virus. However, although the serum antibody levels of these animals following immunization was comparable to that found following infection with live virus, the degree of immunity to infection with virulent influenza virus was measurably less.     

Further studies on subacute encephalitis and hydrocephalus in hamsters caused by measles virus from persistently infected cell cultures

Newborn hamsters were inoculated intracerebrally (IC) with disrupted measles carrier Lu 106 cells. No acute neurological disease developed, but limited, persistent neural infection was identified by immune fluorescence and by virus isolation. By ten days after inoculation, virus could be recovered only by co cultivation of explant cultures of central nervous system (CNS) tissue and Vero cells. Virus was still demonstrable in CNS by both techniques 50 days after inoculation, the latest sample collected. Animals inoculated as newborns developed a poor hem agglutination-inhibiting (HI) antibody response. In three-week-old hamsters inoculated IC no detectable replication of carrier virus occurred. The serum HI antibody response was more than 20 times higher than that in animals inoculated as newborns. Hydrocephalus developed in a fraction of animals inoculated at birth or at the age of three weeks. Both infectious and heat-inactivated disrupted virus carrier material, but not control material of Lu 106 cells, gave hydrocephalus. The precise mechanism leading to hydrocephalus is unclear. Carrier virus material may produce a meningeal irritation causing disturbances of the extraventricular flow or resorption of cerebrospinal fluid, leading to communicating hydrocephalus. Infectious carrier virus may replicate in and destroy ependymal cells, further contributing to hydrocephalus.     

Immunity to Influenza in Ferrets X. Intranasal Immunization of Ferrets with Inactivated Influenza A Virus Vaccines

The response of ferrets after intranasal inoculation of inactivated A/Hong Kong/68 (H3N2) influenza virus vaccines is reported. Normal ferrets given either saline vaccine in drops or freeze-dried vaccine in an aerosol intranasally did not produce detectable serum or nasal hemagglutination inhibiting antibody and were found to be completely susceptible to challenge infection with A/Hong Kong/68 virus. Intranasal saline vaccine did not produce an additive effect on the response of ferrets simultaneously given the same vaccine intramuscularly with adjuvant. Ferrets primed by previous infection with A/PR/8/34 (H0N1) influenza virus, however, responded to intranasal immunization with saline A/Hong Kong/68 virus vaccine and produced serum and nasal antibody. These animals were found to be partially resistant to challenge infection, in contrast to similar animals given saline vaccine intramuscularly which were completely resistant to challenge infection. Primed ferrets did not respond after immunization with the freeze-dried aerosol vaccine, but this may have been due to a failure of the aerosol to be inhaled satisfactorily.     

Immunity to Influenza in Ferrets: I. Response to Live and Killed Virus

Ferrets were found to react with a sharp febrile response to intranasal infection with influenza virus A2/Hong Kong/3/68. Virus was recovered from nasal washings taken 3 days after infection, and virus antibody was found in serum specimens taken 21 days after virus infection. Virus infection produced a pronounced rhinitis; the protein concentration in nasal washings was found to increase three to five-fold with peak levels occurring on day 7, post-infection. Concomitant with the increased protein levels, detectable levels of HI and neutralizing antibody were found in the nasal washings. However, nasal washings taken 13 days or more after influenza virus infection did not contain either increased levels of protein or detectable antibody. These ferrets were immune to re-infection with homologous virus inoculated 5 weeks after primary infection. Thus, ferrets showed no febrile response; virus was not recovered from nasal washings; serum antibody titres did not increase; no increase in protein levels was found in nasal washings; and HI antibody was not found in nasal washings.Using these criteria to assess susceptibility or immunity to influenza virus infection, infection with attenuated influenza virus A2/Hong Kong/1/68 produced immunity to re-infection with virulent virus. Ferrets infected with influenza virus B/England/13/65 or immunized with killed A2/Hong Kong virus did not induce any immunity to infection with influenza virus A2/Hong Kong/3/68.     

Impact of influenza vaccine formulation with a detailed analysis of the cytokine response

Vaccination provides the most effective method of limiting the impact of influenza. Inactivated influenza vaccines are available in three formulations and more information needs to be generated on how antigen presented in different vaccine formulations influences the subsequent immune response. In the present study, we have investigated the effect of two different influenza vaccine formulations on the resulting antibody and cytokine responses and compared these responses with influenza infection. Mice were vaccinated intramuscularly with one or two doses of whole or split virus vaccine or alternatively intranasally infected with influenza virus. Lymphocytes were isolated from spleen cells and stimulated in vitro for 24 or 72 h for analysis of cytokine profile at the gene expression and at the protein level. Additionally, whole blood was collected and the serum antibody response investigated by haemagglutination inhibition (HI) and enzyme-linked immunosorbent assay (ELISA). We found that one dose of whole virus vaccine induced higher antibody and cytokine responses and thus was more immunogenic in unprimed mice than split virus vaccine. Whole virus vaccine induced a strong IFN-gamma (type 1) immune response after one dose of vaccine and a more mixed cytokine response after the second dose. Split virus vaccine induced a type 2 response, particularly after two vaccine doses. Our results show that two doses of vaccine (both vaccine formulation) were more effective in induction of Th2 type of cytokines and thus indicate that both the formulation and also the number of vaccine doses substantially influences the magnitude and quality of the immune response.     

Immunity to influenza in Ferrets

Summary Ferrets inoculated with 300 CCA of inactivated influenza A2/Hong Kong virus vaccine did not produce serum HI antibody, and were completely susceptible to subsequent infection with live A2/Hong Kong virus. Immunization of ferrets with A2/Hong Kong vaccine in Al(OH)₃ induced low levels of serum HI antibody; these animals showed a slightly reduced febrile reaction and reduced titres of virus were recovered from nasal washings following challenge virus infection. Ferrets immunized with inactivated A2/Hong Kong vaccine in Freund's incomplete adjuvant produced relatively high titres of serum HI antibody, but did not produce local antibody detectable in nasal washings. After challenge infection, these animals showed a modified febrile reaction, lower titres of virus were recovered from nasal washings and nasal symptoms were reduced. These results, together with results of similar studies, indicated that the degree of immunity to challenge virus infection was related to the titre of serum HI antibody. However, none of the methods used to induce serum HI antibody gave as solid an immunity as found following live virus infection, although immunization could induce levels of serum HI antibody comparable to that found following virus infection.     

Antibody production and protection against influenza virus in immunodeficient mice.

The roles of T and B cells in the immune response to influenza virus were studied by using mice deficient in either T cells (athymic nude) or immunoglobulin production (CBA/N). The serological responses of these mice to either whole or disrupted A/Aichi/2/68 influenza virus vaccines were examined, and the protective effect of these inoculations was tested by challenge infection with mouse-adapted A/Aichi/2/68 influenza virus. In contrast to normal mice, neither strain of immunodeficient mouse produced detectable serum antibody after inoculation with either type of vaccine. CBA/N mice immunized with intact virus vaccine were protected, however, against subsequent lethal challenge. CBA/N mice inoculated with disrupted virus vaccine and nude mice inoculated with either disrupted or whole virus vaccine were not protected against viral challenge. Evidence of immunological memory was observed in CBA/N and nude mice that had survived live virus challenge after immunization with inactivated vaccine.     

Characterization and long-term persistence of immune response following two doses of an AS03A-adjuvanted H1N1 influenza vaccine in healthy Japanese adults

Background Long-term persistence of immune response and safety of two doses of an A/California/07/2009 H1N1 pandemic influenza vaccine adjuvanted with AS03 (an α-tocopherol oil-in-water emulsion-based Adjuvant System) administered 21 d apart was evaluated in Japanese adults [NCT00989612]. Methods One-hundred healthy subjects aged 20-64 y (stratified [1:1] into two age strata 20-40 y and 41-64 y) received 21 d apart, two doses of AS03-adjuvanted 3.75μg haemagglutinin (HA) H1N1 2009 vaccine. Immunogenicity data by haemagglutination inhibition (HI) assay six months after the first vaccine dose (Day 182) and microneutralization assay following each of the two vaccine doses (Days 21 and 42) and at Day 182 are reported here. Results Persistence of strong HI immune response was observed at Day 182 that met the US and European regulatory thresholds for pandemic influenza vaccines (seroprotection rate: 95%; seroconversion rate: 93%; geometric mean fold-rise: 20). The neutralizing antibody response against the A/Netherlands/602/2009 strain (antigenically similar to vaccine-strain) persisted for at least up to Day 182 (vaccine response rate: 76%; geometric mean titer: 114.4) and paralleled the HI immune response at all time points. No marked difference was observed in HI antibody persistence and neutralising antibody response between the two age strata. The vaccine had a clinically-acceptable safety profile. Conclusion Two priming doses of H1N1 2009 pandemic influenza vaccine induced an immune response persisting for at least six months after the first vaccine dose. This could be beneficial in evaluating the importance and effect of vaccination with this AS03-adjuvanted pandemic influenza vaccine.     

Influenza virus infection of the guinea pig: Immune response and resistance

Guinea pigs were inoculated by intranasal inoculation with unadapted, influenza virus A/England/42/72, and virus was recovered from nasal washings between 3 and 10 days post-inoculation. Infected animals did not exhibit a febrile response to infection, did not produce local antibody and produced only relatively low levels of serum antibody. However, they developed delayed-type hypersensitivity to influenza virus, demonstrable by both skin tests and macrophage migration inhibition tests, which was similar to that of man. The relevance of the influenza virus specific delayed hypersensitivity in immunity to infection was examined in this model. Guinea pigs previously infected with virus or passively immunized with hyperimmune serum were relatively resistant to reinfection with influenza virus A/England/42/72. Inoculation of guinea pigs with spleen cells from immune donor animals, together with or without immune serum, did not give or enhance resistance to challenge virus infection. The results do not suggest a role for delayed hypersensitivity response in immunity to influenza virus infection.     

Humans and Ferrets with Prior H1N1 Influenza Virus Infections Do Not Exhibit Evidence of Original Antigenic Sin after Infection or Vaccination with the 2009 Pandemic H1N1 Influenza Virus

The hypothesis of original antigenic sin (OAS) states that the imprint established by an individual''s first influenza virus infection governs the antibody response thereafter. Subsequent influenza virus infection results in an antibody response against the original infecting virus and an impaired immune response against the newer influenza virus. The purpose of our study was to seek evidence of OAS after infection or vaccination with the 2009 pandemic H1N1 (2009 pH1N1) virus in ferrets and humans previously infected with H1N1 viruses with various antigenic distances from the 2009 pH1N1 virus, including viruses from 1935 through 1999. In ferrets, seasonal H1N1 priming did not diminish the antibody response to infection or vaccination with the 2009 pH1N1 virus, nor did it diminish the T-cell response, indicating the absence of OAS in seasonal H1N1 virus-primed ferrets. Analysis of paired samples of human serum taken before and after vaccination with a monovalent inactivated 2009 pH1N1 vaccine showed a significantly greater-fold rise in the titer of antibody against the 2009 pH1N1 virus than against H1N1 viruses that circulated during the childhood of each subject. Thus, prior experience with H1N1 viruses did not result in an impairment of the antibody response against the 2009 pH1N1 vaccine. Our data from ferrets and humans suggest that prior exposure to H1N1 viruses did not impair the immune response against the 2009 pH1N1 virus.     

Immunogenicity of a monovalent, aluminum-adjuvanted influenza whole virus vaccine for pandemic use

In the case of an influenza pandemic a significant gap between influenza vaccine manufacturing capacities and vaccine demands must be expected on a global scale. This study explores the possibility to increase the number of vaccine doses that can be provided with the existing resources by using a lower amount of antigen per dose in an aluminum-adjuvanted whole virus vaccine formulation, instead of the standard dosage of 15 microg hemagglutinin (HA). The study was performed as an open, non-controlled, randomized, multicentric study in 200 volunteers (18-30 years of age). Three monovalent aluminum-adjuvanted whole virus formulations with different antigen concentrations (1.9, 3.75 and 7.5 microg HA per dose) were compared to a split virus vaccine (15 microg HA per dose) without aluminum adjuvantation. The sera were tested for hemagglutination inhibition (HI) antibodies, neuraminidase inhibition (NI) antibodies and virus neutralizing (VN) antibodies. Nasal swab samples were tested for influenza-specific IgA antibodies. All volunteers were immunologically na?ve to the vaccine strain influenza A/Singapore/1/57 (H2N2). The vaccine was well tolerated. HI titers reached protective levels (geometric mean titer (GMT) >1:40) after two vaccine doses. In the group immunized with the lowest antigen dose the seroprotection rate was 82%. Although the immune response tends to be lower for vaccine formulations with reduced antigen content, the immunogenicity criteria as defined by the European Agency for the Evaluation of Medicinal Products (EMEA) were met with all antigen formulations after two vaccine doses. Significant increases in HI, NI and VN titers were observed, however, no significant local immune response was detected. The use of a low-dose whole virus influenza vaccine, adjuvanted with aluminum appears to be a viable approach to increase vaccine supplies in a pandemic situation.     

Altered antibody response to influenza H1N1 vaccine in healthy elderly people as determined by HI,ELISA, and neutralization assay

To determine the influence of ageing per se as well as of priming histories on the antibody response to influenza vaccination, haemagglutination inhibition (HI), ELISA IgG, IgA, IgM and neutralizing antibody titres were studied in 43 healthy young subjects (mean age 23 years) and 55 healthy elderly people (mean age 79 years). The HI and ELISA IgG responses to the A/Guizhou/54/89 strain (H3N2) for which both the young and the elderly had similar priming histories were equal. By contrast, the HI and IgG responses to A/Taiwan/1/86 (H1N1), where the priming histories were different, were lower in the elderly (P < 0.05). Influenza-specific IgA responses in the elderly tended to be higher for all vaccine strains. Influenza-specific postvaccination IgM titres were similar or tended to be higher in the elderly. A subgroup of elderly subjects (18%) who did not express HI activity to the A/Taiwan/1/86 (H1N1) vaccine strain, reacted in the HI assay with the closely related A/Singapore/6/86 (H1N1) strain. These elderly people, however, produced IgG antibodies which neutralized A/Taiwan/1/86 virus in vitro. It is concluded that the elderly are capable of mounting antibody responses similar to those observed in the young. Moreover, the observed age-related differences in antibody responses to H1N1 strains are probably not due to ageing of the immune system itself, but are determined by differences in priming histories. J. Med. Virol. 55:82–87, 1998. © 1998 Wiley-Liss, Inc.     

Immunogenicity of Fowlpox Virus Expressing the Avian Influenza Virus H5 Gene (TROVAC AIV-H5) in Cats

Vaccination of cats with fowlpox virus expressing the avian influenza (AI) virus H5 hemagglutinin gene (TROVAC AI) resulted in detectable hemagglutination inhibition (HI) antibody responses to the homologous A/Turkey/Ireland/1378/83 (H5N8) (A/tky/Ire/83) AI virus antigen. The HI antibody responses to heterologous A/Chicken/Indonesia/7/03 (H5N1) (A/ck/Indonesia/03) AI virus antigen were also detected in all vaccinated cats, but only after booster vaccinations. The vaccine described in this study and other poxvirus-vectored vaccines may be of value for the prophylaxis of AI virus-associated morbidity and mortality in mammals.     

Influenza infection in ferrets: role of serum antibody in protection and recovery.

The passive administration of ferret antiserum to Ao (H0N1) influenza virus failed to protect the recipient ferrets from subsequent infection with homologous virus. This susceptibility to infection was observed even when the passively acquired serum hemagglutination inhibition (HI) titer was similar to peak convalescent titers. It is therefore concluded that serum antibody alone is probably not a major factor in the prevention of influenza infection. This does not rule out a possible role for serum antibody in prevention of illness. Subsequent to infection, ferrets that had received passive antisera failed to develop high levels of serum HI antibody. In fact, many had no detectable serum antibody (less than 1:8). These animals shed virus for periods of time quite similar to those of infected control animals, which did develope serum antibody. From these data it was concluded that detectable serum HI antibody does not play a significant role in the recovery of ferrets from influenza infection. Interferon was present in high concentrations in the secretions a few days prior to cessation of virus shedding, but it is not clear whether this was the cause of the recovery or merely a concomitant event. Twenty-one days after initial infection two-thirds of the ferrets that had received passive antibody and all control animals were immune to reinfection with the homologous influenza virus. Since the former group had little or no detectable serum HI antibody but most members were immune, there must be some other host mechanism to account for the immunity.     

The immune response to influenza vaccines

Specific immunity to influenza is associated with a systemic immune response (serum haemagglutination inhibition antibody), local respiratory immune response (virus-specific local IgA and IgG antibodies in nasal wash), and with the cell-mediated immune response. Both inactivated and live influenza vaccines induce virus-specific serum antibody which can protect against infection with influenza virus possessing the same antigenic specificity. In the absence of serum antibodies, local antibodies in nasal wash are a major determinant of resistance to infection with influenza virus. In comparative studies in humans it was shown that nasal secretory IgA develops chiefly after immunization with live cold-adapted (CA) vaccine, but persistent nasal secretory IgG was detected in both CA live and inactivated vaccines. The origin of nasal wash haemagglutination inhibition (HI) antibodies is not completely known. Recently it was found that cytotoxic T-cells (CTL) play an important role in immunity against influenza and in clearance of influenza virus from the body. In primed humans, inactivated influenza vaccine stimulates a cross-reactive T-cell response, whereas the ability of inactivated vaccine to stimulate such immunity in unprimed humans has not been determined. Data on the T-cell response to live vaccine in humans are limited to the development of secondary T-cell responses in primed individuals vaccinated with a host-range (HR) attenuated vaccine. The data obtained have shown that immunity induced by inactivated influenza vaccines is presumably dependent on the stimulation of serum antibody. Live CA vaccines not only stimulate a durable serum antibody response, but also induce long-lasting local respiratory tract IgA antibody that plays an important role in host protection.     

Secretory immunological response after intranasal inactivated influenza A virus vaccinations: evidence for immunoglobulin A memory.

An intranasal, inactivated trivalent influenza A vaccine containing 7 micrograms of A/Bangkok/1/79 (H3N2) hemagglutinin was administered to 20 children aged 1 to 6 years to assess the local and systemic immune responses to antigen delivered to the respiratory tract. Six children without prior influenza virus infection exhibited no local immune response and manifested only a minimal systemic response to the intranasal vaccine. In contrast, five individuals who were previously infected with a live attenuated influenza A H3N2 virus vaccine, although having no residual secretory antibody at the time of challenge, promptly developed a local antibody response to intranasal, inactivated antigen. Therefore, the live influenza A virus vaccine had induced memory in the local immunoglobulin A (IgA) immune system. The third group of nine children had previously been infected with wild-type H3N2 influenza virus. A majority of these children had residual local and systemic antibody at the time of challenge but they demonstrated some boosting of local IgA antibody with administration of intranasal inactivated vaccine. The competence of the secretory IgA immune system in young children in mounting primary and secondary responses to influenza antigens has important implications for approaches to prevention of influenzal illness.     

H1亚型流感病毒HA蛋白单克隆抗体的制备及部分特性鉴定

目的:制备针对H1亚型流感病毒HA蛋白的单克隆抗体(mAb),并分析其反应特性。方法:分别以2009年甲型H1N1、季节性A1流感病毒裂解疫苗为免疫原,常规法免疫、融合、克隆化,获得各抗原特异性mAb。应用ELISA、HI试验和Western blot等技术研究mAb的反应性和特异性。结果:获得稳定分泌抗H1亚型流感病毒HA蛋白的杂交瘤细胞97株。其中株特异性mAb39株,29株具有HI活性;亚型特异性mAb7株,5株具有HI活性;2009年流行株与季节性A1、A3流行株共同抗原的mAb16株,9株具有HI活性;针对流感病毒共同抗原mAb35株,22株具有HI活性。结论:两种疫苗均具有较好的免疫原性和免疫保护活性,这些mAb的获得为流感病毒株特异、亚型特异性诊断试剂盒及流感病毒通用诊断试剂盒的制备提供了实验资料,为进一步研究H1N1流感病毒HA的抗原表位奠定了基础。